1. Field of the Invention
The present invention relates to a technique for a microscope, especially a microscope that is preferable for microinsemination, and a microinsemination method using it
2. Description of the Related Art
As one of applications of microscopes, microinsemination in the field of advanced reproductive medicine has been known. The microinsemination is a method of joining sperm and egg under a microscope and fertilizing the egg, which is generally practiced with the Intracytoplasmic sperm injection (ICSI) where a micropipette in which sperm is stored is stabbed into an egg locked by the holding pipette and the sperm is injected directly into the egg. In this method, since the sample is handled on the stage, it is a general practice to use an inverted microscope that has a large operation space above the stage.
Conventionally, in the field of microinsemination, the modulation contrast observation (also called relief contrast observation; hereinafter, referred to as RC observation) has been used. A microscope used for the RC observation is disclosed, for example, in Japanese Laid-open Patent Publication No. 51-29149.
FIG. 15A through FIG. 15C are diagrams for explaining the configuration of the microscope according to the conventional art used for the RC observation. FIG. 15A presents the overall configuration of the microscope used for the RC observation. FIG. 15B is a diagram of a view of the apertured plate included in the condenser lens of the microscope from the optical axis direction. FIG. 15C is a diagram of a view of the modulator included in the objective of the microscope from the optical axis direction.
A microscope 100 illustrated in FIG. 15A includes a polarization plate 101 that rotates around the optical axis, a condenser lens 110, an objective 111 that faces the condenser lens 110 across the sample 106, and an eyepiece 109. The condenser lens 110 includes an apertured plate 103 on which an aperture 102 is formed at a position off the optical axis, a polarization plate 104 that covers a part of the aperture 102, and a lens 105. The objective 111 includes a lens 107, and a modulator 108 that has three areas with a different transmittance (an area 108a having a 100% transmittance, an area 108b having a transmittance of about 25%, and an area 108c having a 0% transmittance). The modulator 108 is positioned on the pupil position of the objective 111, and the modulator 108 and the apertured plate 103 are in an optically-conjugate relationship with each other. Meanwhile, the modulator 108 is only required to be on a position that is optically conjugate with the pupil position of the objective 111. While the modulator 108 is located inside the objective 111 in FIG. 15A, it may also be located on a position that is optically conjugate with the pupil position of the objective 111 outside the objective 111.
According to the RC observation, the egg may be observed three dimensionally by the effect of the oblique illumination due to the aperture 102 and the modulation effect of the modulator 108 on the light through the aperture 102. Therefore, the RC observation is preferable for the ICSI and has been widely used conventionally in the field of microinsemination.
In recent years, in the field of microinsemination, a method of microinsemination in which the microscope is used while switching between a plurality of observation methods as needed to improve the fertility rate has attracted attention. Specifically, a method to use the RC observation mentioned above along with other observation methods such as the differential interference contrast observation (hereinafter, referred to as DIC observation) and polarization observation (hereinafter, referred to as PO observation) is becoming popular.
Since the object may be observed at a high magnification in the DIC observation compared with the RC observation in which a high numerical aperture for illumination may not be obtained, the DIC observation is preferable for observing the sperm that is smaller compared with the egg. For this reason, it is used when selecting a good sperm based on the shape. In addition, the PO observation is preferable for observing the spindle of the egg that has a birefringent property. For this reason, it is used when checking the location of the spindle in order to avoid accidentally damaging the spindle when injecting the sperm into the egg.